Efficient recombinant adeno-associated virus production by a stable rep-cap HeLa cell line correlates with adenovirus-induced amplification of the integrated rep-cap genome

Autor: Anna Salvetti, David Favre, Philippe Moullier, Pascale Nony, Nathalie Provost, Jürgen Kleinschmidt, Jacques Tessier, Gilliane Chadeuf
Přispěvatelé: UMR1089, Laboratoire de Thérapie Génique, Université de Nantes (UN)
Jazyk: angličtina
Rok vydání: 2000
Předmět:
[SDV.BIO]Life Sciences [q-bio]/Biotechnology
viruses
Fluorescent Antibody Technique
MESH: DNA Helicases
MESH: Adenovirus E2 Proteins
MESH: Dependovirus
Virus Replication
medicine.disease_cause
Adenovirus E1B protein
MESH: Adenovirus E1B Proteins
Plasmid
Drug Discovery
MESH: Capsid
Adenovirus E1B Proteins
Adeno-associated virus
MESH: Fluorescent Antibody Technique
Genetics (clinical)
Transfection
Dependovirus
Adenovirus E2 Proteins
DNA-Binding Proteins
MESH: DNA
Recombinant
Molecular Medicine
MESH: Genome
Viral
MESH: Virus Assembly
MESH: Trans-Activators
Blotting
Western
DNA
Recombinant
Genome
Viral
Biology
Cell Line
Viral Proteins
Capsid
Genetics
medicine
Humans
MESH: Blotting
Western
Adenovirus infection
Molecular Biology
MESH: Humans
Virus Assembly
MESH: Transfection
MESH: Virus Replication
DNA Helicases
medicine.disease
Virology
Molecular biology
MESH: Viral Proteins
MESH: Cell Line
Viral replication
Cell culture
MESH: HeLa Cells
Trans-Activators
MESH: DNA-Binding Proteins
HeLa Cells
Zdroj: The Journal of Gene Medicine
The Journal of Gene Medicine, Wiley, 2000, 2 (4), pp.260-268. ⟨10.1002/1521-2254(200007/08)2:43.0.CO;2-8⟩
ISSN: 1099-498X
1521-2254
DOI: 10.1002/1521-2254(200007/08)2:43.0.CO;2-8⟩
Popis: Background A possible procedure for the production of clinical grade recombinant adeno-associated virus type 2 (rAAV) would include the use of packaging cell lines, harboring the rep-cap genes and the vector, combined with a replication defective adenoviral plasmid to provide the helper activities. Several studies have already shown that rAAV can be efficiently assembled by infecting the stable packaging cell line with adenovirus. However, the direct comparison with an adenoviral plasmid has never been reported. Methods To investigate this point, a clone of HeLa and 293 cells harboring one to two rep-cap copies per cell genome (HeRC32 and 293RC21, respectively) were generated. Recombinant AAV was produced by transiently transfecting the AAVCMVLacZ vector and supplying the adenoviral helper activities by either wild-type adenovirus or an adenoviral plasmid (pAdc). As a control, rAAV was similarly produced from naive Hela and 293 cells additionally transfected with a rep-cap plasmid. Results Despite satisfactory rAAV yields from Hela and 293 cells, we show that those from HeRC32 and 293RC21 cells dramatically decrease when adenovirus is replaced by the adenoviral plasmid (pAdc). The analysis performed to identify the factors hampering efficient rAAV assembly by HeRC32 cells in the presence of pAdc shows that: (1) while upon adenovirus infection the integrated rep-cap genome undergoes a dramatic amplification leading to a 100-fold increase in the rep-cap copy number, no amplification is detected upon transfection of pAdc; (2) in pAdc-transfected HeRC32 cells, the intracellular localization of the adenovirus E4orf6 and E1B-55kDa proteins is abnormal as compared to adenovirus-infected cells. Conclusions This study documents that stable rep-cap cells lines are severely hampered for rAAV assembly when a replicative adenovirus is substituted with an adenoviral plasmid. Furthermore, our results also suggest that the lack of amplification of the rep-cap genes, eventually combined with the altered distribution of the adenoviral proteins, E4orf6 and E1B-55kDa, is related to the low rAAV yields observed under these conditions.
Databáze: OpenAIRE
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